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(Circulation. 1996;93:1194-1200.)
© 1996 American Heart Association, Inc.

Articles

Effect of Thrombin Inhibition With Desulfatohirudin on Early Kinetics of Cellular Proliferation After Balloon Angioplasty in Atherosclerotic Rabbits

Michael Ragosta, MD; William L. Barry, MD; Lawrence W. Gimple, MD; S. David Gertz, MD, PhD; Kyle W. McCoy, MD; George A. Stouffer, MD; Coleen A. McNamara, MD; Eric R. Powers, MD; Gary K. Owens, PhD; Ian J. Sarembock, MB, ChB, MD

From the Cardiovascular Division (M.R., W.L.B., L.W.G., K.W.M., G.A.S., C.A.M., E.R.P., I.J.S.), Department of Medicine, and the Department of Molecular Physiology and Cellular Biophysics (G.K.O.), University of Virginia School of Medicine (Charlottesville); and Department of Anatomy and Embryology (S.D.G.), Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Correspondence to Ian J. Sarembock, MD, Box 158, Cardiovascular Division, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail isarembock@virginia.edu.

	Abstract

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Background Thrombin may have a pivotal role in restenosis after angioplasty. Hirudin, a potent thrombin inhibitor, reduces luminal narrowing by plaque after angioplasty in a rabbit model of atherosclerosis. Because cellular proliferation is believed to be an important mechanism for restenosis and thrombin has been shown to be a potent smooth muscle cell mitogen in vitro, we hypothesized that the mechanism of the effect of hirudin on limiting luminal narrowing by plaque occurs via inhibition of cellular proliferation.

Methods and Results Femoral atherosclerosis was induced in 108 rabbits, and balloon angioplasty was performed. At angioplasty, group 1 rabbits (n=38) were treated with a 2-hour infusion of hirudin, and group 2 rabbits (n=41) were treated with heparin. Group 3 rabbits (n=29) were treated with hirudin (n=15) or heparin (n=14) and killed at 7 or 28 days to determine cross-sectional area narrowing by plaque and cellular proliferation with the use of bromodeoxyuridine labeling. At 29, 71, or 167 hours after angioplasty, group 1 and 2 rabbits were injected with ³H-thymidine and killed 1 hour later, and labeling indexes were determined. A significant increase in the index of ³H-thymidine–labeled nuclei was observed in the intima of "ballooned" arteries compared with "nonballooned" atherosclerotic arteries at both 30 hours (0.06±0.05 versus 0.01±0.01, P<.01) and 72 hours (0.10±0.06 versus 0.004±0.004, P<.01). By 7 days, the index of labeled cells was similar to baseline (0.04±0.03 versus 0.01±0.01, P=.12). Hirudin had no effect on the ³H-thymidine labeling indexes at any of the time points studied despite the fact that hirudin treatment in group 3 rabbits resulted in less cross-sectional area narrowing by plaque at both 7 and 28 days after angioplasty (41±16 versus 24±12 at 7 days and 60±21 versus 44±17 at 28 days, heparin versus hirudin; P<.03).

Conclusions Balloon angioplasty resulted in a marked increase in cellular proliferation that peaked at 72 hours. A 2-hour infusion of hirudin failed to reduce early ³H-thymidine labeling, suggesting that inhibition of cell proliferation within the first 7 days after angioplasty is not the predominant mechanism by which hirudin exerts its effect on limiting luminal narrowing by plaque 28 days after balloon angioplasty in this animal model.

Key Words: anticoagulants • atherosclerosis • hirudin • angioplasty

	Introduction

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Atherosclerotic arteries may respond to successful balloon angioplasty with marked renarrowing at the site of dilatation. This process of restenosis is characterized by both cellular and acellular elements. Smooth muscle cells, fibroblasts, and macrophages compose the bulk of the cellular components, whereas extracellular matrix, fibrin, and thrombus compose the acellular elements.^{1 2} Although the precise mechanisms of restenosis are not known, recent data suggest that thrombus and/or the components of the coagulation cascade may have important roles.^{3 4 5 6 7 8} Balloon angioplasty results in arterial wall injury with activation of the coagulation cascade, generation of thrombin, and formation of mural thrombus. Several coagulation factors, including factors X and Xa, protein S, and thrombin, have been shown to be potent smooth muscle cell mitogens in vitro,^{9 10 11 12 13 14} suggesting a potential role for these proteins in the proliferative component of restenosis. We previously demonstrated that 2-hour infusion of the specific thrombin inhibitor recombinant desulfatohirudin resulted in a 50% reduction in luminal narrowing by plaque after balloon angioplasty in an atherosclerotic rabbit model compared with control animals treated with bolus heparin alone.¹⁵ The mechanism of the reduction in luminal narrowing by plaque was not determined in that study.

The proliferative response to arterial injury has been characterized in several animal species, including balloon-withdrawal injury of normal rat carotid¹⁶ and after balloon angioplasty of a carotid plaque created by chronic electrical stimulation in the rabbit.¹⁷ The present study was performed to characterize the early proliferative response after balloon angioplasty in a hypercholesterolemic rabbit model of focal femoral atherosclerosis and to test the hypothesis that potent, early thrombin inhibition with desulfatohirudin reduces restenosis in this model by decreasing cellular proliferation as determined by ³H-thymidine–labeling indexes.

	Methods

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The animal model that we used has been described previously.^{15 18 19}

Induction of Focal Femoral Atherosclerosis
One hundred eight male New Zealand White rabbits (4.0±0.3 kg) were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). Focal atherosclerosis was induced in 10- to 20-mm femoral artery segments by air desiccation endothelial injury, followed by the administration of a 0.02% cholesterol/0.06% peanut oil diet for 28 days, as described previously.^{15 18 19} This diet raised cholesterol levels from 0.93±0.21 mmol/L before cholesterol feeding to 42.6±20.8 mmol/L after 28 days on this diet (P=.0001). The rabbits were housed according to Animal Welfare Act specifications, and all surgical procedures were performed with sterile technique and general anesthesia.

Drug Administration and Experimental Groups
Twenty-eight days after induction of focal femoral atherosclerosis, animals were anesthetized as outlined above and assigned randomly to one of three groups. Fig 1 is a summary of the experimental protocol for group 1 and 2 animals. Group 1 animals (n=38) were treated intravenously with recombinant desulfatohirudin (CGP 39393, CIBA-GEIGY) as a 1.0-mg/kg bolus administered before balloon angioplasty, followed by infusion at 1.0 mg·kg^-1·h^-1 for 2 hours. Group 2 animals (n=41) were treated with a single bolus of intra-arterial heparin (150 units/kg) (heparin sodium injection, 1000 USP units/mL, porcine intestinal mucosa, Solopak Laboratories) before angioplasty to prevent thrombus formation on the catheters. No additional heparin was administered after the bolus dose in these heparin-treated control animals.

View larger version (25K): [in this window] [in a new window]   Figure 1. Flow diagram of experimental protocol. (See text for details.)

These two groups of rabbits (heparin and hirudin treatments) were further divided into three subgroups on the basis of time of death after balloon angioplasty as follows: 30-hour/heparin (16 rabbits), 30-hour/hirudin (13 rabbits), 72-hour/heparin (12 rabbits), 72-hour/hirudin (12 rabbits), 7-day/heparin (13 rabbits), and 7-day/hirudin (13 rabbits).

Because the histological preparation of plastic embedding necessary for thymidine labeling does not allow for the concurrent use of histological stains other than hematoxylin, the effect of hirudin on plaque size 28 days after balloon angioplasty and the effect of hirudin on plaque size and on cellular proliferation 7 days after balloon angioplasty were determined in group 3 rabbits (n=29). Fourteen rabbits were treated with heparin at the time of balloon angioplasty, and 15 rabbits were treated with hirudin as described above. Animals were killed 7 days (heparin: n=4, yielding 7 arteries; hirudin: n=5, yielding 10 arteries) and 28 days (heparin: n=10, yielding 20 arteries; hirudin: n=10, yielding 19 arteries) after balloon angioplasty.

Angiography and Balloon Angioplasty
After right carotid arteriotomy, a 5F Berman catheter (Arrow International) was advanced into the descending aorta and positioned two vertebral spaces above the aortic bifurcation. Baseline angiography of the iliac and femoral arteries was performed 5 minutes after the intra-arterial administration of 1 mL 1% lidocaine with the use of a Siemens Optilux Angiographic System (model 1179878VS048), and images were recorded on 35-mm cineradiographic film with 5 mL diatrizoate meglumine and diatrizoate sodium (Hypaque 76 injection USP, Winthrop-Breon Laboratories) diluted with 5 mL sterile saline. A grid with 10-mm markings was used as an internal calibration standard. The angiographic catheter was replaced by a 0.014-in guide wire, and a 2.5-mm balloon dilatation catheter (Advanced Cardiovascular Systems) was centered across the femoral stenosis under fluoroscopic guidance. Three 60-second, 10-atm balloon inflations were performed at 60-second intervals with the use of a hand inflator. The contralateral femoral stenosis did not undergo balloon angioplasty and served as a "nonballooned" atherosclerotic control. The balloon catheter was replaced by the 5F Berman catheter, and angiography was repeated 10 minutes after the last balloon inflation. The catheter and vascular sheath were removed, the carotid artery was ligated, and the wound was closed. Group 1 and 2 rabbits were kept alive for 30 hours, 72 hours, or 7 days (168 hours) after balloon angioplasty, whereas group 3 rabbits were kept alive for 7 days or 28 days after balloon angioplasty. All three groups were fed normal rabbit chow after balloon angioplasty. Final angiography was performed just before death, with the left carotid artery as described above.

Blood Samples
Activated partial thromboplastin time (aPTT) was determined before drug administration, after angioplasty, and at the conclusion of the 2-hour drug infusion in the first 18 consecutive hirudin-treated rabbits (group 1) and the first 8 consecutive heparin-treated rabbits (group 2). Cholesterol levels were measured with rabbits in a fasting state before initiation of the high cholesterol diet and 28 days after high cholesterol feeding.

³H-Thymidine Autoradiography, Determination of Luminal Narrowing, and Bromodeoxyuridine Staining
At 29, 71, or 167 hours after balloon angioplasty (1 hour before death), group 1 and 2 rabbits were injected intraperitoneally with 1 mCi/kg ³H-thymidine (1 µCi/mL, 6.7 Ci/mmol, New England Nuclear). These time points were studied because they span the time period in which maximum proliferation rates occur in other vascular injury models.^{16 17} One hour after administration of ³H-thymidine, the final angiogram was performed and the animals were killed with an overdose of pentobarbital sodium. The distal aorta and iliac arteries were perfused at physiological pressure (100 mm Hg and 22°C) with 100 mL of 0.016% glutaraldehyde in phosphate buffer (Polysciences). The femoral artery segments (40 to 50 mm) were excised bilaterally, with the proximal and distal ends marked with silk sutures. Specimens were trimmed and placed in 0.025% glutaraldehyde for 3 hours. Specimens were then washed in PBS (137 mmol/L NaCl, 8.1 mmol/L Na₂HPO₄, 2.7 mmol/L KCl, 1.5 mmol/L KH₂PO₄, pH 7.4), dehydrated with ethyl alcohol, and embedded in JB-4 (Polysciences). The trimmed, excised femoral arterial segments measuring 10 mm in length were cut into cross sections at 3-mm intervals, yielding three 3-mm segments. These segments were further sectioned at 5-µm intervals with a dry glass knife and mounted onto slides. The slides were coated with NTB-2 emulsion (Eastman Kodak), diluted 1:1 with distilled water, stored at 4°C for 2 weeks, developed (D19, Eastman Kodak), fixed (Rapid-Fix, Eastman Kodak), and stained with hematoxylin (Sigma Chemical Co).

The limits of the neointima and media were determined by identifying the internal and external elastic laminae under both regular and phase light microscopy. Because portions of the laminae were not visible in some vessels, the neointimal and medial boundaries were estimated on the basis of morphological distinctions between these zones. When the neointimal and medial boundaries could not be estimated, the neointima and media were counted together. Labeling indexes were determined for each section by counting cells under oil immersion. A micrometer was moved circumferentially around the vessel, and contiguous but not overlapping portions of the vessel were sampled. The number of labeled nuclei and total nuclei within the limits of the micrometer field were recorded, and a labeling index was determined by dividing the labeled cells by the total number of cells. The labeling index indicates the proportion of labeled cells expressed as a decimal (eg, 6 labeled cells in a total of 100 cells is a labeling index of 0.06).

Group 3 rabbits were killed 7 days (n=9) and 28 days (n=20) after balloon angioplasty with an overdose of pentobarbital sodium. One hour before death, 7-day rabbits were injected intraperitoneally with 30 mg/kg bromodeoxyuridine (Sigma), a thymidine analogue. Bromodeoxyuridine solutions were prepared in sterile 0.5 mol/L Na₂CO₃/NaHCO₃ buffer, pH 9.8. After pressure perfusion at physiological pressure with glutaraldehyde as described above, femoral arterial segments were excised, dehydrated in ethanol and xylene, and embedded in paraffin. Serial 5-µm sections from each arterial segment were stained with hematoxylin and eosin to determine luminal narrowing, and bromodeoxyuridine positivity was detected in proliferating cells with the use of a monoclonal antibody against bromodeoxyuridine (mouse IgG₁, DAKO) in sequential sections from animals killed 7 days after angioplasty. Cross-sectional area narrowing by plaque was determined in both the 7-day and 28-day rabbits by measuring the luminal area and the area subscribed by the internal elastic lamina with a computer-assisted method previously described.¹⁵ Cellular proliferation indexes were measured by counting the number of bromodeoxyuridine-positive cells per 1000 µm of plaque area.

Angiography
Angiograms were analyzed quantitatively with a computer-assisted technique described previously.^{18 19 20} Observers were blinded to treatment group and time after angioplasty. The minimum luminal diameters (in mm) at the site of focal femoral artery stenoses and adjacent segments proximal and distal to the stenoses were determined with the aid of a computer-generated line placed perpendicular to the long axis of the artery. Templates were drawn of the preangioplasty angiogram to ensure that subsequent analysis was performed on the same arterial segments.

Statistical Analysis
Data are reported as the number of femoral arteries in each group, and angiographic, ³H-thymidine–labeling, cross-sectional area narrowing by plaque and bromodeoxyuridine-labeling data are expressed as mean±SD. Angiographic data and proliferation indexes were analyzed with one-way ANOVA and Student's t test to evaluate two-tailed levels of significance. Paired and unpaired tests were used as appropriate. For values that were not normally distributed, a Mann-Whitney U test was used. Comparison of categorical data was made with the two-tailed Fisher's exact test.

	Results

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aPTTs
The mean aPTT at baseline for all animals was 76±28 seconds. Ten minutes after drug bolus, the aPTT increased 2.4±0.9 times baseline in the hirudin-treated animals and 2.6±0.2 times baseline in the heparin-treated animals (P=NS, heparin versus hirudin). At the completion of and at 2 hours after balloon angioplasty, the aPTT remained greater than twice baseline in all heparin-treated control and hirudin-treated animals (2.2±0.5 times baseline for hirudin-treated animals and 2.8±0.1 times baseline for heparin-treated animals).

Angiographic Data
Quantitative angiography was performed on the first 10 consecutive animals at each time point in group 1 and 2 animals. Arteries that were occluded at death (one each in the 72-hour/hirudin, 7-day/hirudin, and 7-day/heparin groups) were excluded from angiographic analysis. The mean minimal luminal diameters before, immediately after, and at 30, 72, and 168 hours after angioplasty in 29 heparin-treated control arteries and 27 hirudin-treated arteries are summarized in Table 1. The mean minimum luminal diameter increased significantly after balloon angioplasty in all groups (P<.001). Although we have previously shown a significant decrease in luminal diameter in heparin-treated control animals 28 days after balloon angioplasty,^{15 18 19} no decreases were discerned by angiography at the three early time points examined in the present study. Likewise, no significant differences in luminal diameter were observed between hirudin-treated arteries and heparin-treated control animals either before or immediately after angioplasty or at 30, 72, or 168 hours after angioplasty.

View this table: [in this window] [in a new window]   Table 1. Angiographic Assessment of Luminal Diameter

³H-Thymidine Incorporation
³H-Thymidine autoradiography was performed on 79 ballooned arteries and 16 contralateral, nonballooned atherosclerotic control arteries. A representative section from a ballooned femoral artery illustrating the ³H-thymidine–labeling technique used in the study is shown in Fig 2. The internal elastic lamina was identified in all except 11 ballooned arteries (14 of 16 30-hour/heparin-treated control arteries, 12 of 13 30-hour/hirudin-treated arteries, 8 of 12 72-hour/heparin-treated control arteries, 8 of 12 72-hour/hirudin-treated arteries, 13 of 13 168-hour/heparin-treated control arteries, and 13 of 13 168-hour/hirudin-treated arteries). In the 11 ballooned arteries in which the internal elastic lamina was not clearly visualized, no attempt was made to assign ³H-thymidine-labeling indexes to either the intima or media. In these arteries, the percentage of labeled cells was determined for the intima plus media combined.

View larger version (126K): [in this window] [in a new window]   Figure 2. Representative photomicrograph of a femoral arterial segment after balloon angioplasty. Nuclei labeled with 3H-thymidine are shown in the intima (I) and media (M) (straight arrows). iel indicates internal elastic lamina; L, lumen. Hematoxylin stain, oil immersion.

Time Course of Cellular Proliferation and Effect of Balloon Angioplasty
Thymidine labeling indexes were determined at 30, 72, and 168 hours in the ballooned and nonballooned arteries for both heparin-treated control and hirudin-treated arteries. Fewer than 0.01 of the cells were labeled with ³H-thymidine at each of the three time points studied in nonballooned arteries (Table 2). Balloon angioplasty resulted in a significant increase in the mean ³H-thymidine-labeling index in the intima, with a 6-fold increase in the mean index of labeled cells at 30 hours (0.06±0.05 versus 0.01±0.01, P<.01) and a >20-fold increase at 72 hours (0.10±0.06 versus 0.004±0.004, P<.01). At 168 hours after angioplasty, the index of intima-labeled cells decreased toward baseline (0.04±0.03 for ballooned versus 0.01±0.01 for nonballooned, P=.12). Similar changes in both the magnitude and kinetics of ³H-thymidine labeling were observed in the media alone and in the intima plus media combined.

View this table: [in this window] [in a new window]   Table 2. 3H-Thymidine Labeling Indexes

Effect of Hirudin on Cellular Proliferation
Fig 3 depicts the index of cells labeled with ³H-thymidine in the intima (Fig 3A), media (Fig 3B), and intima plus media combined (Fig 3C) for individual arteries at each of the three time points. At 30 hours, the index of labeled cells in the intima was 0.06±0.05 for heparin-treated control animals and 0.06±0.07 for hirudin-treated animals (P=.96). At 72 hours, the ³H-thymidine-labeling index in the intima was 0.09±0.04 versus 0.11±0.08 for heparin-treated control and hirudin-treated animals, respectively (P=.53). Seven days (168 hours) after angioplasty, 0.04±0.03 of cells in the intima of heparin-treated control animals and 0.04±0.04 of cells in the intima of hirudin-treated animals were labeled with ³H-thymidine (P=.77). Thus, no significant difference in labeling index was observed between the hirudin-treated and heparin-treated vessels at any of the three time points studied with respect to the intima, the media, or the intima plus media combined.

View larger version (12K): [in this window] [in a new window]   Figure 3. Plots of 3H-thymidine labeling indexes in the intima (A), media (B), and combined intima plus media (C) 30, 72, and 168 hours after balloon angioplasty in hirudin- () and heparin- () treated animals.

Histomorphometric Analysis and Early Cellular Proliferation With Bromodeoxyuridine Labeling
Luminal area, area bounded by the internal elastic lamina, and cross-sectional area narrowing by plaque were determined in group 3 rabbits 7 days and 28 days after balloon angioplasty; cellular proliferation with bromodeoxyuridine labeling was assessed in nine rabbits (heparin, n=4; hirudin, n=5) 7 days after angioplasty (Table 3). The area bounded by the internal elastic lamina was not significantly different between the heparin-treated and hirudin-treated rabbits at both 7 and 28 days after angioplasty. The luminal area tended to be smaller in the heparin-treated rabbits at 7 days and was significantly smaller in the heparin-treated rabbits at 28 days (643±313 versus 1075±737 x10³·µm^-2). Consistent with our earlier report,¹⁵ the percent cross-sectional area narrowing by plaque 28 days after balloon angioplasty was found to be significantly less in hirudin-treated rabbits than in heparin-treated control animals (44±17% versus 60±21%, P=.009). This beneficial effect of hirudin on cross-sectional area narrowing by plaque was observed early, with significantly less cross-sectional narrowing by plaque seen in the hirudin group 7 days after balloon angioplasty (24±12% versus 41±16%, P=.03). Importantly, serial sections for bromodeoxyuridine labeling demonstrated no difference in the number of cells positive for bromodeoxyuridine per 1000 µm of plaque area in hirudin-treated versus heparin-treated rabbits 7 days after balloon angioplasty (0.078±0.084 versus 0.103±0.098, P=.57). This was noted despite the observed differences in percent cross-sectional area narrowing by plaque between heparin- and hirudin-treated groups at this early time point after balloon angioplasty.

View this table: [in this window] [in a new window]   Table 3. Bromodeoxyuridine Labeling Index and Histomorphometric Analysis

	Discussion

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The major objectives of the present study were to determine the time course of cellular proliferation in the intima and media of atherosclerotic femoral arteries in hypercholesterolemic rabbits after balloon angioplasty and to determine whether hirudin inhibits cellular proliferation, thus providing a plausible mechanism by which short-term (2-hour) infusion of hirudin at the time of balloon dilatation reduces restenosis in this animal model.¹⁵ Balloon angioplasty induced a significant increase in cellular proliferation in both the intima and media during the first 7 days after angioplasty, being highest at 72 hours. However, hirudin treatment was not associated with a reduction in early cellular proliferation despite a significant reduction in plaque size at both 7 and 28 days.

The mechanisms responsible for restenosis remain poorly understood and are thought to result from a complex interaction of platelet-rich thrombus formation, release of vasoactive and mitogenic factors, migration and proliferation of smooth muscle cells and possibly macrophages in the intima of the dilated arterial segment, elaboration of extracellular matrix, and arterial remodeling.⁴ Thrombin, a multifunctional serine protease, is generated in large amounts at the site of arterial wall injury and is known to regulate hemostasis and thrombosis.^{21 22} Animal studies have shown that thrombin inhibitors, particularly hirudin, play a pivotal role in preventing acute platelet-rich thrombosis after deep arterial injury.^{23 24 25 26} Numerous additional thrombin-mediated cellular functions that may have an impact on restenosis include a role in smooth muscle cell mitogenesis and as a chemotactic factor for inflammatory cells, including macrophages.^{27 28} Although the critical function of thrombin in hemostasis and thrombosis has been established, the in vivo significance of its proliferative and inflammatory actions remains to be defined.

There is considerable evidence that smooth muscle cell proliferation is important in the development of a neointima after injury of normal arteries. Although the molecular and cellular responses to balloon injury of normal arteries (first injury) have been characterized by others,^{16 29} the arterial responses after balloon angioplasty of atherosclerotic arteries (second injury) are less well described. In a rabbit carotid artery model in which weak electrical stimulation is used to induce fibromuscular atheroma, followed by balloon angioplasty (a second-injury model), peak proliferative activity occurs in the intima at 3 days, with later and less-marked proliferation in the media.¹⁷ These results suggest that balloon injury of a preexisting plaque (second injury) initiates smooth muscle cell proliferation predominantly in the intima. Also, later proliferation in the media (at 21 days) in this rabbit carotid model may be important for lesion growth. The present study characterizes the growth response of cells within atherosclerotic plaque of hypercholesterolemic rabbits and confirms that intimal proliferation occurs early, being highest at 72 hours, but, in addition, demonstrates significant proliferation in the media at these early time points, with a reduction in the labeling index in both the intima and the media by 7 days.

Cellular proliferation in human lesions after balloon angioplasty is difficult to study systematically and is limited to examination of atherosclerotic coronary arteries from hearts removed at the time of cardiac transplantation or tissue obtained at the time of directional atherectomy.^{30 31 32 33} Several studies have used an explant method of cellular outgrowth or cellular proliferation by immunocytochemical staining for proliferating cell nuclear antigen. The kinetics of cellular outgrowth is higher in smooth muscle cells from restenotic compared with primary lesions.^{31 32 33} However, another study found very low levels of cellular proliferation by proliferating cell nuclear antigen staining, suggesting that mechanisms other than proliferation may also be important.³⁴ However, very few specimens were obtained at early time points after the initial intervention. In addition, the use of local antiproliferative therapy (methotrexate by local delivery) did not lower restenosis in an animal model.³⁵ Thus, the contribution of cellular proliferation to the restenosis process remains unclear.

It is unlikely that methodological limitations account for the findings in our study. Because a single pulse of ³H-thymidine 1 hour before death was used (rather than continuous labeling), it is possible that a small but biologically significant effect on labeling indexes would not be detected.³⁶ Also, results of the present study do not rule out the possibility that hirudin inhibited proliferation of a specific cell type or subpopulation of cells in the lesion, since cell-specific markers were not used. It is unlikely that we missed a transient hirudin effect on cellular proliferation during the initial 7 days after balloon angioplasty, particularly since the numbers of vessels analyzed were large and P values approached 1 at the time points examined. Although it is possible that hirudin had a delayed effect on labeling indexes that was evident only beyond 7 days, this seems unlikely since labeling indexes in both heparin-treated control and hirudin-treated groups had decreased by 1 week to values similar to those for nonballooned control animals. Also, a significant effect on cross-sectional narrowing by plaque was already evident in the hirudin-treated animals by 7 days after balloon angioplasty. Thus, the most likely interpretation of these data is that hirudin reduces cross-sectional area narrowing by plaque by a mechanism other than inhibition of cellular proliferation in this animal model. Alternative explanations include (1) that hirudin inhibits cellular migration rather than proliferation^{27 28} ; (2) that hirudin reduces mural thrombosis, resulting in less thrombus incorporation into the plaque; and (3) other, as-yet-undescribed effects of hirudin, including limiting matrix production; modulating growth factor, chemokine, and adhesion molecule expression and production; and limiting foam cell accumulation. Some of these mechanisms are under investigation.

Finally, although hirudin resulted in smaller cross-sectional area narrowing by plaque after balloon angioplasty compared with heparin-treated rabbits, no difference in luminal diameter by angiography was seen at early time points between heparin- and hirudin-treated animals. It is likely that small differences in luminal diameter between hirudin- and heparin-treated rabbits exist at these early time points but are beyond the limit of resolution of the quantitative angiographic system used in this and earlier studies (0.1 mm).

In conclusion, balloon angioplasty of atherosclerotic femoral arteries in hypercholesterolemic rabbits resulted in a significant increase in cellular proliferation in both the intima and media, being highest at 72 hours after arterial injury. With the technique of cell labeling with a single pulse of ³H-thymidine 1 hour before death, no difference in cellular proliferation indexes was detected between heparin-treated control animals and hirudin-treated animals despite significantly less cross-sectional area narrowing by plaque at both 7 and 28 days after balloon angioplasty in hirudin-treated animals. This suggests that inhibition of cellular proliferation is not the predominant mechanism by which hirudin exerts its beneficial effect on limiting cross-sectional narrowing by plaque in this model.

	Acknowledgments

 
This study was supported in part by Public Health Service grant R01-HL-47849 from the National Institutes of Health (Dr Sarembock), American Heart Association Grant-in-Aid (VA-92-GIA) (Dr Sarembock), and Fellowship Grant (VA-91-F48) from the American Heart Association, Virginia Affiliate (Dr Ragosta). The authors thank the staff of the vivarium of the Department of Comparative Medicine at the University of Virginia for their excellent animal care. We also thank John F. DeFrancisco, James Uyeki, Thao Anh Tu, and Leewei Ang for expert technical assistance.

Received July 12, 1995; revision received October 18, 1995; accepted October 23, 1995.

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